Despite decades of research on post-translational modification enzymes, little is known about their dynamic, spatiotemporal activities on a subcellular level?and yet the nature of these subcellular activities is often key to understanding their dysfunction in cancer. The goal of this project is to develop a fluorescence lifetime-based technology to map those activities in living cells at subcellular resolution. This approach is based on robust preliminary data in which we demonstrate feasibility for detecting tyrosine kinase activity (an important PTM in many cancers) via highly resolved fluorescence lifetime shifts for three peptide probes (for Abl, Src-family, and Syk kinases), including multiplexed analysis of two probes at a time. In Aim 1, we will fully characterize the physiochemical mechanism that gives rise to the shift, which involves binding of the phosphorylated peptide product of the kinase reaction to an SH2 domain. This will allow us to understand and predict lifetime shift effects, enabling targeted probe design. In Aim 2, we will characterize and further develop the multiplexed analysis of multiple probes, using a biologically relevant model system (the integrin signaling pathway) to validate the utility of the method. In Aim 3, we will broaden the scope of the technology to detect other PTM enzyme activities, starting with Ser/Thr kinases and acetyltransferases. The work described in this proposal will deliver 1) a suite of novel, complementary probes for PTM enzyme function in cells, 2) a strategy to develop additional probes, 3) high-throughput in vitro methods for detecting PTMs via fluorescence lifetime shifts, and 4) cutting edge methods for imaging multiple probes in the same cell. These tools will make unique contributions to the understanding of PTM dynamics in the cell and to the elucidation of roles for subcellular kinase signaling in disease.